The invention concerns an improved pyrogenicity test for use in automated immunoassay systems, in which a sample is incubated with a monocyte-containing reagent in a pyrogen-free assay system comprising a surface preferably coated with pyrogen-free anti-cytokine antibodies. The invention also concerns pyrogen-free assay systems coated with pyrogen-free antibodies for use in these tests. The invention also relates to a test to measure the basal production of endogenous mediators of the inflammatory response by leukocytes and a method of measuring the capacity of a leukocyte to respond to a stimuli.
When certain chemical or biological compounds are brought into contact with the circulatory system of humans or other mammals, they cause a systemic response characterized by an elevation in body temperature, or fever. Such materials are called xe2x80x9cpyrogensxe2x80x9d or xe2x80x9cpyrogenicxe2x80x9d compounds. Compounds which pose a particular risk of pyrogenicity include medical products which can be inhaled, injected or infused, and medical devices such as membranes or implanted materials. Even nutrients can represent a risk of pyrogenicity. In addition to the pyrogenic nature of the product itself or by-products of its production, bacterial contamination of the product can often cause pyrogenicity. This problem persists even if the product is xe2x80x9csterilizedxe2x80x9d by heat or chemical methods, as the main pyrogenic component of the bacteria, endotoxin (or cell wall lipopolysaccharide), can remain after the bacteria are killed.
Usually, compounds which act as a pyrogen do so by stimulating cytokine production in monocytes after contact with tissue, cells or body fluids. It is these endogenously produced cytokines which produce the fever response in the affected organism. The most important known fever producing cytokines are the proteins interleukin-1 (IL-1), interleukin-1ra (IL-1ra), interleukin-6 (IL-6), interleukin-8 (IL-8) and tumor necrosis factor (TNF), as well as low molecular weight lipid mediators such as prostaglandin E2 (PGE2). These compounds are routinely assayed by ELISA, or enzyme-linked immunosorbent assays (for IL-1, IL6, or TNF), and EIA, or enzyme immunoassay (for PGE2).
In order to avoid a pyrogenic reaction and ensure the safety of any drug or pharmaceutical product administered parenterally, pyrogenic contamination must be monitored to identify individual lots that are contaminated with bacteria. Two pharmacopial methods, the Limulus amebocyte lysate (LAL) test and the rabbit pyrogen test, are currently used routinely to monitor pyrogen contamination in mass-produced pharmaceutical products.
The rabbit test is an in-vivo test which consists of injecting a statistically significant number of rabbits with the compound, and observing the average rise in body temperature elicited in the test animals. Although the rabbit test is responsive to a wide spectrum of pyrogenic agents, including bacterial endotoxin, the rabbit test has a relatively low sensitivity (ng endotoxin/ml) compared to other pyrogen tests (pg endotoxin/ml for the LAL test). In addition, the correlation between species of pyrogenic responses to compounds is, at best, rough. It has been documented, for instance, that the dose of bacterial endotoxin eliciting a pyrogenic response varies as much as 10,000 times between species. Rabbit tests have the further disadvantage of taking one to two days to administer properly. The expense, relative insensitivity, and ethical issues involved in animal testing have made the rabbit test disfavored in recent years.
Among the compounds causing fever, one of the best described is endotoxin (lipopolysaccharide, LPS), which comes from the bacterial wall of Gram-negative germs (Moltz et al., Neurosci. Biobehav. Rev., 1993, 17, 237-269; Tilders et al., Psychoneuroendocrinology, 1994, 19, 209-232; Rothwell, Crit. Rev. Neurobiol., 1994, 8, 1-10; Zeisberger and Roth, Neuropsychobiology, 1993, 28, 106-109). It was, therefore, thought to be generally useful to replace expensive and time consuming rabbit experiments with a direct LAL test for endotoxin. This approach has obvious limitations. The LAL test is a very sensitive in-vitro test; however, it only detects endotoxins from Gram-negative bacteria and gives false negative results with certain products which can still stimulate monocytes to make pyrogenic cytokines. The LAL test is also altered by endotoxin-binding components that are present in blood or blood components (Harris et al, J. Lab. Clin. Med., 1991, 118, 186-193; Emancipator et al., 1992, Infect. Immun., 60, 596-601; Read et al., Eur. Heart J., 1993, 14, 125-129). Some of these endotoxin-binding components bind to LPS and prevent it from being detected. These components may also affect the immune reaction with monocytes, i.e., the primary pyrogenic reaction. This interference is problematic, as testing for exogenous pyrogens in blood products is essential in order to ensure safe administration of these products in the clinical setting. On the other hand, the Limulus test is so sensitive that it is easily prone to false positive results due to impurities that are not relevant to product quality (Fujiwara et al., Yakugaku Zasshi, 1990, 110, 332-340).
It has been observed that cytokines are produced by whole blood when endotoxin is added, ex vivo. After incubation of whole blood with Sal. minnesota lipopolysaccharide for six hours, an increase in IL-1 xcex2 and TNF-xcex1 could be detected in the culture by ELISA. (M. B. Finch, et al., xe2x80x9cCytokine Production In Whole Blood ex vivo,xe2x80x9d Agents and Actions 34:49-52 (1991), C. E. Desch, et al., xe2x80x98Production of Human Tumor Necrosis Factor from Whole Blood Ex Vivo,xe2x80x9d Lymphokine Rsrch, 8:141-46 (1989)). In addition, cultured monocyte cell lines have also been observed to produce IL-1 xcex2 and IL-6 when incubated with E. coli endotoxin, and it has been suggested that such cell lines can be used as an effective pyrogen test.(Taktak et al., xe2x80x9cAssay of Pyrogens by Interleukin-6 Release from Monocytic Cell Lines,xe2x80x9d J. Pharm. Pharmacol. 143:578-582 (1991)).
U.S. Pat. No. 5,891,728 describes a method of bringing a sample of potentially pyrogenic material into contact with human whole blood to release cytokines from monocytes and other white blood cells. An advantage of this procedure is that the use of a complete biological reagent (whole blood) allowed assessment of the exposure of humans and other mammals to pyrogenic agents in the test sample. All blood components that are needed for an interaction of the exogenous pyrogen with leukocytes (e.g., LPS binding proteinxe2x80x94LBP, soluble CD 14, etc.) are present. After incubation of the sample with whole blood in a test container, the incubation mixture is centrifuged, clarified, and assayed for the presence of secreted cytokines in a separate immunoassay step.
Although this test method is an improvement over the LAL test, and potentially detects a large variety of potential exogenous pyrogens other than endotoxin, it has several disadvantages which prevent or severely limit its use on an industrial scale. The two separate steps of culturing the blood with test material and assaying the culture for cytokine production are done in separate containers, requiring the cultured material to be transferred to a new container for immunoassay. The transfer of material necessitated by these separate steps is not easily done utilizing modem quality control assay equipment. Pyrogen tests are normally (and preferably) carried out simultaneously, testing numerous lots of pharmaceutical products for pyrogen contamination. Thus, a need exists for a pyrogenicity test which can be carried out in a single, assay container system, and which combines the steps of sample incubation with the pyrogen-detecting biological reagent and the capture of cytokine(s) produced by the biological reagent.
Applicants have developed an in-vitro pyrogen test that is very sensitive, detects any pyrogens capable of producing a fever response (i.e. endotoxins from Gram-negative and Gram-positive bacteria and non-lipopolysaccharide materials), and is inexpensive. Generally, the present invention is drawn to a method of detecting pyrogens in a sample by incubating the sample with a monocyte-containing reagent in a pyrogen-free assay system comprising a surface coated with pyrogen-free anti-cytokine antibodies, and assaying the assay system for the presence of cytokines bound to the surface by the coating antibodies.
In preferred embodiments of the present invention, the monocyte-containing reagent comprises whole blood, such as whole human blood. The monocyte-containing reagent may further comprise an anticoagulant or a diluent or both.
In preferred embodiments of the pyrogen test of the present invention, the cytokine assayed for in the assay system is interleukin-1, interleukin-1ra, interleukin-6, interleukin-8, tumor necrosis factor-xcex1, or prostaglandin E2.
In preferred embodiments of the pyrogen test of the present invention, the assay system is a microtiter well, and the surface on which the antibody is coated is a portion of the interior of the microtiter well.
In preferred embodiments of the pyrogen test of the present invention, the pyrogen-free assay system is assayed by a calorimetric enzyme-linked immunosorbent assay for cytokine bound to the anti-cytokine antibody coated on the surface of the assay system.
The present invention is also drawn to a pyrogen-free assay system comprising a surface coated with pyrogen-free anti-cytokine antibodies. In preferred embodiments of the invention, the assay system is a microtiter well wherein a pyrogen-free antibody to a cytokine is coated on a portion of the interior of the microtiter well. In more preferred embodiments, the well is part of a planar array of similar wells, situated so that the array of wells may be read with automated immunoassay plate reading equipment. In preferred embodiments of assay system of the present invention, the cytokine to which the antibodies bind is interleukin-1, interleukin-1ra, interleukin-6, interleukin-8, tumor necrosis factor-xcex1, or prostaglandin E2.